Process for the culturing of cells

ABSTRACT

The invention relates to a process for the culturing of cells, preferably E1-immortalized HER cells, more preferably PER.C6 cells in a reactor in suspension in a cell culture medium, wherein the cells produce a biological substance, preferably an antibody, wherein at least one cell culture medium component is fed to the cell culture and wherein the cell culture comprising the cells, the biological substance and cell culture medium is circulated over a separation system and wherein the separation system separates the biological substance from substances having a lower molecular weight than the biological substance and wherein the biological substance is retained in or fed back into the reactor. Preferably part of the substances of lower molecular weight is continuously removed from the cell culture.

This application is the U.S. national phase of International ApplicationNo. PCT/EP2007/005915, filed 4 Jul. 2007, which designated the U.S. andclaims priority to Europe Application No(s). 06014671.9, filed 14 Jul.2006 and 07002571.3, filed 6Feb. 2007, the entire contents of each ofwhich are hereby incorporated by reference.

The invention relates to a process for the culturing of cells in areactor in suspension in a cell culture medium.

Such a process is for example known from WO04/099396. Herein it isdescribed how the cell density of the cell culture and the yield of thedesired biological material can be improved by optimizing the growthconditions in a fed-batch process.

Furthermore, WO05/095578 discloses a process for the culturing of cellsby continuous perfusion culturing of a cell culture comprising cellculture medium and cells, wherein cell culture medium is added to thecell culture, the cell culture is circulated over a filter modulecomprising hollow fibers resulting in an outflow of liquid having alower cell density than the cell culture, and the flow within the filtermodule is an alternating tangential flow, wherein the cells produce abiological substance. In the examples of WO05/095578 it is shown that0.9 g/L/day product is produced, corresponding to a productconcentration in the outflow of approximately 0.3 g/L.

The larger the volume of liquid containing the biological substance, themore laborious becomes the purification of the biological substance. Theconcentration of biological substance obtained is not that high in theprocesses as disclosed in WO04/099396 and WO05/095578. Therefore,downstream processing of this biological substance is cumbersome,because the biological substance needs to be concentrated before furtherpurification steps are applied or large volumes of less concentratedbiological substance need to be purified. Furthermore, the culturing ofcells at lower cell densities results in lower volumetric productivityand therefore requires larger and/or more culturing vessels and thushigher investments in equipment for a given production level.

Therefore, it is the object of the invention to provide a processwherein the product is obtained from the cell culture in higherconcentrations.

A further object of the present invention is to enable culturing of thecells and production of the biological material during an extendedperiod.

These objects are achieved by a process for the culturing of cells in areactor in suspension in a cell culture medium, wherein the cellsproduce a biological substance, wherein at least one cell culture mediumcomponent is fed to the cell culture and wherein the cell culturecomprising the biological substance and cell culture is circulated overa separation system and wherein the separation system separates thebiological substance from substances having a lower molecular weightthan the biological substance and wherein the biological substance isretained in or fed back into the reactor.

For example, the invention relates to a process for the culturing ofcells in a reactor in a cell culture medium, wherein the cells produce abiological substance, wherein nutrients and/or cell culture mediumis/are fed to the reactor and wherein the cell culture comprising thecells and the cell culture medium is circulated over a filter having apore size or molecular weight cut off of between 5 and 500 kD.

It has been found that by using a separation system that separates thebiological substance from substances having a lower molecular weightthan the biological substance, the biological substance can beaccumulated in the cell culture in higher concentrations.

Hence, the present invention differs form the cell culturing describedin the prior art in that it allows for accumulation of the desiredbiological material together with the cell mass.

In a preferred embodiment of the present invention part of thesubstances of lower molecular weight are continuously removed from thecell culture.

An additional advantage of the process of the present invention is thathigher viable cell concentration can be reached as compared to forexample batch or fed-batch processes. Furthermore, the productiontime—the period during which the cells produce the biologicalsubstance—can be extended compared to for example batch or fed-batchprocesses. Also, as compared to a batch or fed-batch process, it ispossible to use a smaller reactor. Use of smaller reactors is ofadvantage as this reduces the equipment and facility relatedinvestments.

Also, higher concentrations of the biological substance may be obtainedin shorter times.

It was found that it was possible to obtain high concentrations ofbiological substance within the reactor without sharply decreasing thecell viability and hence without limiting the production time. Theperson skilled in the art would have expected that the productinhibition, i.e. inhibition of production of the biological substance bythe biological substance itself or inhibition by other macromoleculesproduced by the cell (such as for instance host cell proteins, enzymesor cellular debris), would occur. Furthermore, it was found that theaccumulation of the desired biological material does not impair thefunction of the separation system.

The process of the present invention provides a considerable advantagein terms of cell density, product concentration in the cell culture andextended culturing period as compared to the processes according toWO05/095578 and WO04/099396. As a result the present process results inan improved production of the desired biological material.

Cells which can be used to produce the biological substance are inprinciple all cells known to the person skilled in the art, which havethe ability to produce a biological product. The cells may beeukaryotic, for example, filamentous fungi, for example Aspergillusniger, Aspergillus oryzae, Trichoderma reesei, Penicillium chrysogenum,yeasts, for example Saccharomyces cerevisiae, Kluyveromyces lactis,Phaffia rhodozyma, yeast from the genus Pichia, for example Pichiapastoris or prokaryotic, for instance Escherichia coli, Bacillus sp, forexample B. licheniformis, B. subtilis, B. amyloliquefaciens, B.alkalophilus, Streptomyces sp., Corynebacterium glutamicum, Pseudomonassp. Examples of eukaryotic cells are for example also described in Chu,L., Robinson, D. K., (2001) Curr. Opinion Biotechn., vol. 12, p.180-187. Preferably, the cells that are used in the process of thepresent invention are animal cells, in particular mammalian cells.Examples of mammalian cells include CHO (Chinese Hamster Ovary) cells,hybridomas, BHK (Baby Hamster Kidney) cells, myeloma cells, human cells,for example HEK-293 cells, human lymphoblastoid cells, E1 immortalizedHER cells, mouse cells, for example NS0 cells. More preferably, E1immortalized HER cells are used, most preferably PER.C6 cells.

Primary human embryonic retina (HER) cells can be isolated from fetuses(Byrd P, Brown K W, Gallimore P H. 1982. Malignant transformation ofhuman embryo retinoblasts by cloned adenovirus 12 DNA. Nature 298:69-71, Byrd P J, Grand R J A, Gallimore P H. 1988. Differentialtransformation of primary human embryo retinal cells by adenovirus E1regions and combinations of E1A+ras. Oncogene 2: 477-484). Primary cellswill die upon culturing for several passages. E1-immortalized HER cellsfor the purpose of the present invention are derived from primary HERcells by expressing DNA encoding adenoviral E1A and E1B proteinstherein, to obtain immortalized cells. Such immortalized cells can becultured for more than 100 passages. Methods to obtain E1-immortalizedHER cells have for instance been described in U.S. Pat. No. 5,994,128,in Byrd P, Brown K W, Gallimore P H. 1982. Malignant transformation ofhuman embryo retinoblasts by cloned adenovirus 12 DNA. Nature 298:69-71, in Byrd P J, Grand R J A, Gallimore P H. 1988. Differentialtransformation of primary human embryo retinal cells by adenovirus E1regions and combinations of E1A +ras. Oncogene 2: 477-484, and inGallimore, P.H., Grand, R. J. A. and Byrd, P. J. (1986). Transformationof human embryo retinoblasts with simian virus 40, adenovirus and rasoncogenes. AntiCancer Res. 6, p499-508. For instance, immortalized HERcells, including PER.C1, PER.C3, PER.C4, PER.C5, PER.C6, PER.C8 andPER.C9 cells, were generated by transfection of primary HER cells usinga plasmid that contained the adenovirus serotype 5 (Ad5) E1A- andE1B-coding sequences (Ad5 nucleotides 459-3510) under the control of thehuman phosphoglycerate kinase (“PGK”) promoter (see U.S. Pat. No.5,994,128).

In a preferred embodiment, the cells in the process of the presentinvention are E1-immortalized HER cells, more preferably PER.C6 cells(see U.S. Pat. No. 5,994,128). PER.C6 cells are exemplified by cells asdeposited under ECACC No. 96022940 (see, e.g., U.S. Pat. No. 5,994,128,EP 0833934 B1).

In the process of the invention, the cells may be cultured in suspensionin any form, for example as immobilized cells, single cells or in cellclusters or as a combination thereof. Preferably the cells are culturedas single cells and/or as small cell clusters of not more than 100cells, more preferably of not more than 20 cells. Cells can for examplebe immobilized on microcarriers such as are commercially available fromfor example GE Healthcare (Cytodex).

A reactor as defined herein is a system that comprises the cell culturewhich cell culture on its turn comprises cells and a cell culturemedium. It preferably provides sterile barriers, such as air filters, toprevent other cells from contaminating the desired cells and itpreferably maintains a favorable environment for the cells by providingthe right culture conditions such as mixing, temperature, pH, oxygenconcentration etc.

The reactor can for example be of a more permanent nature, for examplethe reactor can be of stainless steel or glass or can for example be ofa disposable nature, for example the reactor can be a plastic flask orbag. Examples of reactors suitable for use in the present inventioninclude, but are not limited to stirred tank vessels, airlift vesselsand disposable bags that can be mixed by rocking, shaking motion orstirring. Preferably disposable (bio)reactors are used as they arefavourable as they require relatively low investment costs, have greatoperational flexibility, short turn-around times and are easilyconfigurable to the process. Disposable (bio)reactors are commerciallyavailable from for example Hyclone, Sartorius, Applikon or Wave.

The term ‘separation system’ is defined within the framework of theinvention as a system capable of separating on basis of molecularweight. The separation system used in the process of the invention iscapable of separating the biological substance from substances having alower molecular weight than the biological substance. In other words,the molecular weight cut-off is chosen such that the molecular weightcut-off (MWCO) is smaller than, more preferably at least a factor 2,most preferably at least a factor 3 smaller than the molecular weight ofthe biological substance. Typically, but of course depending on themolecular weight of the biological substance produced in the process ofthe present invention, the MWCO of the separation system is preferablyat least 5, more preferably at least 10, most preferably at least 30 kDaand preferably at most 500 kDa, more preferably at most 300 kDa, mostpreferably at most 100 kDa. For example for an IgG with a molecularweight of 150 kDa, a separation system having a MWCO of at most 50 kDais most preferred.

Examples of separation systems include but are not limited to filters,centrifuges and aqueous two phase extraction systems.

The term ‘filter’ as used herein is meant to include all devices withthe ability to separate particles on basis of size or molecular weight.In principle, in the process of the present invention, any filter may beused as long as the pore size or MWCO is chosen such that the biologicalsubstance is separated from substances having a lower molecular weightthan the biological substance, typically this will be a pore size orMWCO of between 5 and 500 kDa. Examples of filters suitable for use inthe present invention include membrane filters, ceramic filters andmetal filters. The filter may be used in any shape; the filter may forexample be spiral wound or tubular or may be used in the form of asheet. Preferably, in the process of the invention, the filter used is amembrane filter, preferably a hollow fiber filter. With the term “hollowfiber” is meant a tubular membrane. The internal diameter of the tube isat least 0.1 mm, more preferably at least 0.5 mm, most preferably atleast 0.75 mm and preferably the internal diameter of the tube is atmost 10 mm, more preferably at most 6 mm, most preferably at most 1 mm.Filter modules comprising hollow fibers are commercially available fromfor example General Electric (GE, formerly Amersham).

By circulating the cell culture comprising the biological substance,cells and the cell culture medium over a separation system, thebiological substance and cells are retained in the reactor and theliquid outflow therefore has a lower concentration of biologicalsubstance and a lower cell density than the cell culture. Usually in theprocess of the invention, the liquid outflow does not contain or hardlycontains any biological substance and cells. Usually, the liquid outflowwill essentially only contain components having a molecular weight lowerthan that of the biological substance. Essentially all cells andessentially all biological substance are therefore usually retained inthe reactor.

Preferably, the pore size or MWCO of the filter is chosen such that thesize of the pores or MWCO of the filter is smaller than, more preferablyat least a factor 2, most preferably at least a factor 3 smaller thanthe diameter or molecular weight of the product, ensuring a highretention of product. Typically, but of course depending on the size ormolecular weight of the product, i.e. biological substance produced inthe process of the present invention, the pore size or MWCO of thefilter is preferably at least 5, more preferably at least 10, mostpreferably at least 30 kDa and/or the pore size or MWCO of thefilter/membrane is preferably at most 500 kDa, more preferably at most300 kDa, most preferably at most 100 kDa.

With molecular weight cut-off (MWCO) is meant the molecular weight abovewhich at least 90% of the particles is retained by the separationsystem.

Circulating the cell culture over a separation system, for example afilter means that the cell culture is passed through a separationsystem, for example a filter resulting in a liquid outflow and a flowwhich contents are kept in or fed back into the reactor. The flow whichcontents are kept in or fed back into the reactor will usuallyessentially only contain components having a molecular weight of atleast equal to that of the biological substance or higher and thereforesaid flow will comprise more biological substance than the liquidoutflow.

In principle, it is not critical when the circulation of the cellculture over the separation system is started during the process of theinvention. The circulation of the cell culture may for example bestarted directly from the start of the process or when the viable celldensity of the cells has reached a certain level.

The circulation of the cell culture over a filter may be a flowsubstantially perpendicular with respect to the filter surface, alsoknown as dead-end flow or a flow substantially parallel to the filtersurface, also known as tangential flow, for example unidirectionaltangential flow (TFF) or cross-flow. A preferred example of cross-flowis alternating tangential flow (ATF) as with ATF it was found thatfilter clogging does not occur (quickly) even at very high celldensities. It is common general knowledge that in depth filtration, thefinal small pore filter needs to be protected from clogging by coursepre-filters. This practice is based on the common general knowledge thatfilters with smaller pores or with a smaller MWCO clog more easily,thereby limiting the production time. If ATF is used, the use of apre-filter becomes superfluous.

The flow may be directed by moving the cell culture, by moving thefilter or both. The filter may for example be moved by rotation(rotating filter) or vibration (vibrating filter). Alternatively if theflow is directed by moving the cell culture only, the filter is staticand the cell culture may for example be moved by way of pumps orpressure.

With “alternating tangential flow” is meant that there is one flow inthe same direction as (i.e. tangential to) the filter surface(s), whichflow is going back and forth, and that there is another flow in adirection substantially perpendicular to said filter surface.Alternating tangential flow can be achieved according to methods knownto the person skilled in the art (for example as described in U.S. Pat.No. 6,544,424).

During the culturing of the cells, at least one cell culture mediumcomponent, for example one or more nutrients and/or cell culture mediummay be fed to the cells. In the process according to the invention, itis of advantage to supplement in part or preferably in whole at leastone of the depleted nutrients by way of a feed of this nutrient or thesenutrients to the reactor. For example, complete cell culture medium maybe fed to the reactor, which is of advantage as a separate feed needthen not be prepared separately. The cell culture medium may for examplealso be fed to the cells in a more concentrated form; this is ofadvantage as smaller volumes are easier to handle. Also one or morenutrients may be fed to the reactor. For example carbohydrates, forinstance glucose or fructose; amino acids, such as glutamine and/orpeptides may advantageously be fed to the reactor.

In a preferred embodiment of the invention, cell culture conditions arechosen such that cell growth rate and/or specific productivity of thecells is not limited and more preferably such that the concentration ofat least one of the components of the cell culture medium remainsessentially constant. Examples of limiting cell culture conditions arenutrient limitations and formation of inhibiting metabolites, such asammonia, carbon dioxide and lactate. For example, cell cultureconditions such as the feed may be chosen such that cell growth rate isnot limited for example by supplying enough nutrients as to compensatefor the depletion and/or to avoid the production of inhibitingmetabolites such as lactate or ammonia. For example, the aerationconditions may be chosen such that carbon dioxide formation is notlimiting the cell growth rate. Growing the cell under non-limitingconditions is highly advantageous from a Good Manufacturing Practice(GMP) point of view as 1) this may give a constant cell cultureenvironment that in many cases also gives constant and good productquality and 2) this may lead to high cell viability, in some cases to acell viability of more than 98%. High cell viability reduces the releaseof cell related contaminants, such as host cell proteins, whichfacilitates product purification. Furthermore, growing the cells atunlimited cell growth rate and/or unlimited specific productivity hasthe commercial advantage that it is possible to produce more biologicalsubstance in an even shorter time as higher cell density will be reachedearlier in the process.

“Specific productivity” of the cells is the amount of a given biologicalsubstance produced per cell per time unit and is usually expressed inpg. cell⁻¹day⁻¹.

The rate of addition of at least one cell culture medium component, forexample nutrients and/or cell culture medium to the cell culture (theinflow rate or perfusion rate) influences the viability and the densityof the cells. In the process of the invention, the cell culture mediumcomponent(s), such as nutrients and/or cell culture medium may be fedfor example in a continuous flow, semi-continuous flow, for examplestep-wise flow or staggered flow. Preferably, cell culture mediumcomponent(s), for example nutrients and/or cell culture medium are addedin a continuous flow.

Cell culture medium component(s), such as complete cell culture mediumand/or nutrients may in principle be fed to the reactor at any timeduring the process. Preferably, the feed is initiated before substrates,such as glutamine and glucose have reached such low levels as to causethe growth of the cells to cease or before inhibitory metabolites, forexample lactate or, ammonia reach such high levels that growth wouldcease. From this point onward, the cell culture medium component(s),such as nutrients and/or complete cell culture medium are preferably fedto the reactor at a rate such that substrate demand is met.

In one embodiment of the invention, cell culture medium is added at aFeed Rate according to formula (1):Feed Rate=SFR×(total cell culture volume)×(viable cell density)  (1)wherein the feed rate is expressed in liters per day, wherein the SFR isthe Specific Feed Rate, i.e. the rate in which the cell culture mediumis fed to the cell culture expressed as the volume of medium added perviable cell per time unit and wherein the viable cell density is thenumber of viable cells per unit of volume. The number of viable cellscan be determined by the person skilled in the art, for example via thetrypan blue exclusion method. The specific feed rate is preferablychosen between 0.01 and 0.3 nL/cell/day, more preferably between 0.01and 0.2 nL/cell/day.

It may be of advantage to take into account additional parameters whenadjusting the feed rate, for example the amount of glucose to be fed tothe culture and/or the oxygen uptake rate. For example, for PER.C6 thefeed rate of the cell culture medium and/or the nutrients is preferablychosen such that glucose concentration is kept between 3 and 20 mmol/L,more preferably between 5 and 15 mmol/L. Preferably the glucoseconcentration is at least 3 mmol/L, more preferably at least 5 mmol/Land preferably at most 20 mmol/L, more preferably at most 15 mmol/L.

In a special embodiment of the invention cell culture (comprising cells,biological substance and cell culture medium) is removed at least oncefrom the reactor and liquid, for example cell culture medium or anutrient feed is added to the reactor to compensate for the cell cultureremoval. Cell culture removal may lead to longer process times at highcell densities in combination with high cell viabilities resulting in ahigher productivity. Cell culture may be removed continuously orstep-wise.

In a preferred embodiment of the invention, cell culture (comprisingcells, cell culture medium and biological substance) is removed from thereactor as soon as the desired cell density, for example a cell densityof at least 10.10⁶ viable cells/ml, preferably of at least 20.10⁶ viablecells/ml, more preferably of at least 30.10⁶ viable cells/ml, forexample a cell density of at most 200. 10⁶ viable cells/ml, is reachedand liquid, for example cell culture medium or nutrient feed is added tothe reactor to compensate for the cell culture removal. Preferably, cellculture is removed at such rate that the cell density remains in thedesired cell density range. This embodiment of the invention is highlyadvantageous as compared to a conventional batch or fed-batch process asit combines the advantages of the process of the invention with highviability that can be maintained longer, making it possible to realizean even higher overall volumetric productivity. With ‘volumetricproductivity’ is meant the amount of biological substance produced perunit reactor volume per unit time and is usually expressed ing.L⁻¹.day⁻¹. As compared to a conventional perfusion process, thisembodiment of the invention is also highly advantageous as it combinesthe advantages of the process of the invention with a cell cultureremoval stream having a high concentration of biological substance. Thehigh concentration of biological substance in the cell culture removalstream makes it commercially interesting to harvest the biologicalsubstance there from. In a conventional perfusion process wherein cellculture is removed, the cell culture removal stream does not containenough biological substance to make it commercially worthwhile toharvest the biological substance and the cell culture removal stream istherefore usually regarded as waste. Hence, in this embodiment of theinvention, in theory all biological substance produced can be harvestedin a straight forward, economically feasible and simple manner.

In a particularly preferred embodiment of the invention, cell cultureconditions are chosen such that the cell growth rate and/or specificproductivity of the cells is not limited and more preferably such thatalso the concentration of at least one of the components of the cellculture medium, such as glucose or glutamine remains constant and cellculture is removed at least once from the reactor as soon as the desiredcell density is reached and liquid, for example cell culture medium isadded to the reactor to compensate for the cell culture removal.

Preferably, the rate of the outflow is chosen such that it issubstantially equal to the rate of the addition of the at least one cellculture medium component, for example nutrients and/or cell culturemedium minus the rate of the optional cell culture removal.

Cells that produce a biological substance are for instance cells capableof expressing a gene encoding the biological substance. Cells capable ofexpressing a gene encoding the biological substance may for example beprepared by transfection of the cells with a plasmid containing the geneencoding the biological substance and gene encoding a suitable selectionmarker, for example a gene encoding a neomycine resistance (Neo markergene). Stably transfected cells may then be selected by selectionpressure, for example—in the case of a Neo marker gene—by culturing thetransfected cells in the presence of G418 (genericin) and immediatescreening of the cells for cells exhibiting high-level expression of thebiological substance. Methods for preparing clones of E1-immortalizedHER cells expressing a protein, and methods for culturing such cells toproduce the protein, are well known to the skilled person, and can forinstance be found in U.S. Pat. No. 6,855,544.

Biological substances, which may be produced by the cells, for exampleby expressing a (recombinant) gene coding therefore are for example(recombinant) proteins, in particular receptors, enzymes, fusionproteins, blood proteins such as proteins from the blood coagulationcascade, multifunctional proteins such as for instance erythropoietin,virus or bacterial proteins for instance for use in vaccines;immunoglobulins such as antibodies, for example IgG or IgM, and thelike; Preferably a protein, more preferably an antibody is produced bythe cells. Preferably, the biological substances such as proteins orvaccines produced by the cells can be used as an active ingredient in apharmaceutical preparation. In the context of the present invention, theterms ‘product’ and ‘biological substance’ are interchangeable.

Within the framework of the present invention, with pharmaceuticalpreparation is meant any preparation, which can be used as a medicine,in particular as a medicine in humans. Such a medicine may for examplebe used for diagnosis, or for prophylactic purpose such as for instancea vaccine, and/or for therapeutic purpose, such as for instance anenzyme or protein for which a patient is deficient, or an antibody tokill undesired cells. A pharmaceutical preparation may further contain apharmaceutically acceptable carrier or excipient, examples of which arewell known to the person skilled in the art.

The PER.C6 cell line can be used for production of biologicalsubstances, such as E1-deleted adenovirus (see e.g. U.S. Pat. No.6,994,128; Nichols et al, 2002, Propagation of adenoviral vectors: useof PER.C6 cells. In: Curiel D, Douglas J T, editors. Adenoviral vectorsfor gene therapy. San Diego: Elsevier. p 129-167), other viruses (seee.g. WO 01/38362), or recombinant proteins (see e.g. U.S. Pat. No.6,855,544; Yallop et al, 2005, PER.C6 cells for the manufacture ofbiopharmaceutical proteins, Modern Biopharmaceuticals: Design,Development and Optimization, 4 Volumes, 779-807, Jörg Knäblein(Editor)).

Examples of proteins that can be used as an active ingredient inpharmaceutical preparations (with the brand name between brackets)include Tenecteplase (TN Kase™), (recombinant) antihemophilic factor(ReFacto™), lymphoblastoid Interferon α-n1 (Wellferon™), (recombinant)Coagulation factor (NovoSeven™), Etanercept, (Enbrel™), Trastuzumab(Herceptin™), Infliximab (Remicade™), Palivizumab (Synagis™),Basiliximab (Simulect™), Daclizumab (Zenapaz™), Rituximab (Rituxan™),(recombinant) Coagulation factor IX (Benefix™) and Interferon β-1a(Avonex™).

Examples of vaccines that can be used as an active ingredient inpharmaceutical preparation include isolated protein antigens, examplesof which include but are not limited to live, oral, tetravalentRotavirus vaccine (RotaShield™), rabies vaccine (RanAvert™), influenzavaccines and inactivated hepatitis A vaccine (VAQTA™).

The pH, temperature, dissolved oxygen concentration and osmolarity ofthe cell culture medium are in principle not critical and depend on thetype of cell chosen. Preferably, the pH, temperature, dissolved oxygenconcentration and osmolarity are chosen such that it is optimal for thegrowth and productivity of the cells. The person skilled in the artknows how to find the optimal pH, temperature, dissolved oxygenconcentration and osmolarity for the culture (see. e.g. WO 2004/099396).Preferably, for the process of the invention when using E1 immortalizedHER cells, the pH is chosen between 6.6 and 7.6 and/or the temperatureis chosen between 30 and 39° C. and/or the osmolarity is chosen between260 and 400 mOsm/kg. To maintain optimal process conditions automationto control the process conditions is desired. In order to optimizeprocess conditions, for instance to obtain growth arrest for increasedcellular productivity, during the culture a shift in the cultureconditions can be applied. This may be established by for instance atemperature shift (such as from 37 to 32° C.), a pH shift or anosmolarity shift.

The process of the present invention can in principle be performed inany type of cell culture medium suitable for the culturing of cells.Guidelines for choosing a cell culture medium and cell cultureconditions are well known and are for instance provided in Chapter 8 and9 of Freshney, R. I. Culture of animal cells (a manual of basictechniques), 4th edition 2000, Wiley-Liss and in Doyle, A., Griffiths,J. B., Newell, D. G. Cell & Tissue culture: Laboratory Procedures 1993,John Wiley & Sons.

For example the cell culture medium may for example comprise as a cellculture medium component a carbohydrate source, salts and/or amino acidsand/or vitamins and/or lipids and/or detergents and/or buffers and/orgrowth factors and/or hormones and/or cytokines and/or trace elements.Examples of carbohydrate sources include glucose, fructose, galactoseand pyruvate. Examples of salts include magnesium salts, for exampleMgCl₂.6H₂O, MgSO₄ and MgSO₄.7H₂O iron salts, for example FeSO₄.7H₂O,potassium salts, for example KH₂PO₄, KCl; sodium salts, for exampleNaH₂PO₄, Na₂HPO₄ and calcium salts, for example CaCl₂.2H₂O. Examples ofamino acids include all known proteinogenic amino acids, for examplehystidine, glutamine, threonine, serine, methionine. Examples ofvitamins include: ascorbate, biotin, choline.Cl, myo-inositol,D-panthothenate, riboflavin. Examples of lipids include: fatty acids,for example linoleic acid and oleic acid; Examples of detergents includeTween® 80 and Pluronic® F68. Example of buffers include HEPES andNa₂CO₃. Examples of growth factors/hormones/cytokines include IGF(insulin-like growth factor), hydrocortisone and (recombinant) insulin.Examples of trace elements are known to the person skilled in the artand include Zn, Mg and Se. The cell culture medium may for example alsocomprise other cell culture medium components, for example soy peptoneor ethanol amine.

For production of biological substances according to the invention, inparticular if the biological substances are to be used as an activeingredient in pharmaceutical preparations, serum free media arepreferred to media containing a serum source. The reason for this isthat serum source media may be contaminated with viruses, present therisk of prionic infections, and can create a major obstacle in thedownstream processing of the biopharmaceutical product (i.e. the furtherpurification of the biological substance from the cell culture).Therefore the process of the invention is preferably performed in a cellculture medium that does not comprise serum from an animal, includinghuman, source. Since compounds from a mammalian source also present aninfection risk, preferably the cell culture medium is mammalian sourcefree (i.e. the cell culture medium does not comprise serum or componentsfrom a mammalian source). More preferably the cell culture medium isanimal source free (i.e. the cell culture medium does not comprise serumor components from an animal, including human, source. Examples of serumfree media that can be used for the culturing of PER.C6 cells includecommercially available media, such as for instance EX Cell™ VPRO medium(SAFC), HyQ® CDM4Retino™ (HyClone), IS ProVec CD (Irvine scientific),293-SFM II (invitrogen).

In preferred embodiments, the biological substance produced in theprocess of the present invention is harvested from the flow whichcontents are kept in or preferably fed back into the reactor or from thecell culture that is removed from the reactor or from both. Thebiological substance(s) produced in the process of the present inventioncan be further harvested from the cell culture in so-called downstreamprocessing, using methods dependent on the biological substance, whichmethods are as such well known to the skilled person. Downstreamprocessing usually comprises several purification steps in varyingcombinations and order. Examples of purification steps in the downstreamprocessing are separation steps (e.g. by affinity chromatography and/orion exchange chromatography and/or extraction by aqueous two-phasesystems and/or precipitation by for example ammonium sulphate), stepsfor the concentration of the biological substance (e.g. byultrafiltration or diafiltration), steps to exchange buffers and/orsteps to remove or inactivate viruses (e.g. by virus filtration, pHshift or solvent detergent treatment).

In one aspect, the invention relates to a cell culture comprisingmammalian cells, preferably E1-immortalized HER cells, more preferablyPER.C6 cells, having a viable cell density of at least 50.10⁶ cells/mL,preferably at least 60.10⁶ cells/mL, in particular at least 90.10⁶cells/mL and a concentration of biological substance of at least 5 g/L,more preferably at least 10 g/L, in particular at least 11 g/L. Inprinciple the concentration of biological substance can be as high asthe solubility of the biological substance allows. The concentration ofviable cells is typically not more than 200.10⁶ cellslmL and preferablywithin the range of 80-150.10⁶ cells/mL.

Viable cell density can for example be determined using the tryptan blueexclusion method for example by using a cell counter as is commerciallyavailable from for example Innovatis (Cedex cell counter).

With cell culture is meant the liquid comprising cell culture medium,cells and biological substance, which liquid is the result of a processfor the culturing of cells in a reactor in a cell culture medium,wherein the cells produce the biological substance.

The invention will now be elucidated by way of the following exampleswithout however being limited thereto.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the viable cell density Y (10⁶.ml⁻¹) plotted versus theprocess time X (days) for process A (batch), B (fed-batch) and C1(process of the invention).

FIG. 2 shows the IgG concentration in the reactor Z (% as compared toIgG concentration in process A) versus the process time X (days)forprocess A (batch), B (fed-batch) and C1 (process of the invention).

FIG. 3 shows the viable cell density Y (10⁶.ml⁻¹) plotted versus theprocess time X (days) for process A (batch), B (fed-batch) C2 (processof the invention).

FIG. 4 shows the IgG concentration in the reactor Z (% as compared toIgG concentration in process A) versus the process time X (days) forprocess A (batch), B (fed-batch) and C2 (process of the invention).

FIG. 5 shows the viable cell density Y (10⁶.ml⁻¹) plotted versus theprocess time X (days) for process A (batch), B (fed-batch) C3 (processof the invention).

FIG. 6 shows the IgG concentration in the reactor Z (% as compared toIgG concentration in process A3) versus the process time X (days) forprocess A, and C3.

FIG. 7 shows the cumulative yield Q (% as compared to yield in processA, per L reactor volume) plotted versus the process time X (days) forprocess A, B and C3.

FIG. 8 shows the cell number Y (10⁶.ml⁻¹) plotted versus the processtime X (days) for C4 (process of the invention).

FIG. 9 shows the IgG concentration in the reactor Z (% as compared tothe maximum IgG concentration reached versus the process time X (days)for process C4 (one embodiment of process of the invention)

EXAMPLES Example 1 Comparison Between a Batch Process, a Fed BatchProcess and the Process According to the Invention

In this example the performance of the process according to the presentinvention was compared to batch and fed-batch processes.

FIG. 1 shows the viable cell density Y (10⁶.ml⁻¹) plotted versus theprocess time X (days) for process A (batch), B (fed-batch) and C1(process of the invention).

FIG. 2 shows the IgG concentration in the reactor Z (% as compared toIgG concentration in process A) versus the process time X (days)forprocess A (batch), B (fed-batch) and C1 (process of the invention).

All fermentations were performed using a Sartorius Biostat B controllerto control the temperature at 36.5° C., the pH between 7.2 and 6.8 andthe DO at 50% air saturation and at 200 rpm. The same IgG producingPER.C6 cell line (see WO 2004/099396) was used in all experiments.

Batch process A

The batch process was executed at 4 L working volume in a Sartorius B5vessel. Cells were inoculated at 3×10e5 cells/mL in VPRO medium (SAFC)supplemented with 6 mM L-glutamin and subsequently cultured for 17 days.

Fed-Batch process B

The fed-batch process was executed at 4 L working volume in a SartoriusB5 vessel. Cells were inoculated at 3×10e5 cells/mL in VPRO medium(SAFC) supplemented with 6 mM L-Glutamin. During the culture glucose andglutamine were added to keep the concentration above respectively 15 mMand 1 mM, Amino acids and peptides were added from day 5 to replenishthe consumed amino acids.

Process of the Invention C1

The process of the invention was performed in a 2 L Applikon vessel. A100 kDa Molecular Weight Cut-Off (MWCO) hollow fiber membrane obtainedfrom General Electric (GE) operated in ATF flow mode with an ATF-2system (Refine Technology) was used to retain the cells and the IgGproduct. The culture was started with 3×10e5 cells/mL in VPRO medium(SAFC) supplemented with 6 mM L-Glutamin. VPRO culture medium (SAFC)supplemented with 6 mM L-Glutamin was perfused through the suspensioncell culture using a Specific Flow Rate (SFR) between 0.05 and 0.2nL/cell/day. The highest product concentration obtained was 1.4 g/L.

The process of the invention resulted in increased viable cell densitiesand increased product concentrations compared to mentioned cultivationmodes in less time, as can be seen from FIG. 1 and FIG. 2 below.

Example 2 Comparison Between a Batch Process, a Fed Batch Process andthe Process According to the Invention

In this example the process according to the present invention is againcompared to batch and fed-batch processes; in process C2, the CO₂pressure is controlled and a 50 kDa separation system was used.

FIG. 3 shows the viable cell density Y (10⁶.ml⁻¹) plotted versus theprocess time X (days) for process A (batch), B (fed-batch) C2 (processof the invention).

FIG. 4 shows the IgG concentration in the reactor Z (% as compared toIgG concentration in process A) versus the process time X (days) forprocess A (batch), B (fed-batch) and C2 (process of the invention)

All fermentations were performed using a Sartorius Biostat B controllerto control temperature at 36.5° C., pH between 7.2 and 6.8 and DO at 50%air saturation and at 200 rpm. The same IgG (of approximately 150 kDa)producing PER.C6 cell line (see WO 2004/099396) was used in allexperiments.

Batch Process A

The batch process was executed at 4 L working volume in a Sartorius B5vessel. Cells were inoculated at 3.10⁵ cells.mL⁻¹ in VPRO medium (SAFC)supplemented with 6 mM L-glutamin and subsequently cultured for 17 days.

Fed-Batch Process B

The fed-Batch process was executed at 4 L working volume in a SartoriusB5 vessel. Cells were inoculated at 3.10⁵ cells.mL⁻¹ in VPRO medium(SAFC) supplemented with 6 mM L-Glutamin. During the culture glucose andglutamine were added to keep the concentration above respectively 15 mMand 1 mM. Amino Acids and peptides were added from day 5 to replenishthe consumed amino acids.

Process of the Invention C2

The process of the invention was performed in a 2 L Applikon vessel. A50 kDa Molecular Weight Cut-Off (MWCO) hollow fiber membrane (GE)operated in ATF flow mode with an ATF-2 system (Refine Technology) wasused to retain the cells and the IgG product. The culture was startedwith 3×10e5 cells/mL in VPRO medium (SAFC) supplemented with 6 mML-Glutamin. VPRO culture medium (SAFC) supplemented with 6 mM L-Glutaminis perfused through the suspension cell culture using an SPR between0.05 and 0.2 nL.cell⁻¹.day⁻¹. The CO₂ pressure was controlled below 15%.

Result

As can be seen from FIG. 3 and from FIG. 4, the process according to theinvention results in significantly increased viable cell densities andincreased product concentrations (2415%×Batch yield; 690%×Fed-Batchyield) in equal or less time (100% Batch time; 81% Fed-Batch time).

The overall productivity increase in g.L⁻¹.day⁻¹ of the process of theinvention is 23.9 times the Batch productivity in g.L⁻¹.day⁻¹) and 8.5times the Fed-batch productivity in g.L⁻¹. day⁻¹. In the process of theinvention C2, 11.1 g product/L was produced. Clogging of the retentiondevice did not occur during 17 days, even with very high cell density.

Example 3 Comparison Between a Batch Process, a Fed Batch Process andthe Process According to the Invention

In this example the performance of the process according to the presentinvention with cell culture removal and again compared to batch andfed-batch processes; in process C3 cell culture has been removed.

FIG. 5 shows the viable cell density Y (10⁶.ml⁻¹) plotted versus theprocess time X (days) for process A (batch), B (fed-batch) C3 (processof the invention).

FIG. 6 shows the IgG concentration in the reactor Z (% as compared toIgG concentration in process A3) versus the process time X (days) forprocess A, and C3.

FIG. 7 shows the cumulative yield Q (% as compared to yield in processA, per L reactor volume) plotted versus the process time X (days) forprocess A, B and C3.

All fermentations were performed using a Sartorius Biostat B controllerto control temperature at 36.5° C., pH between 7.2 and 6.8 and DO at 50%air saturation and at 200 rpm. The same IgG (of approximately 150 kDa)producing PER.C6 cell line (see WO 2004/099396) was used in allexperiments.

Batch Process A

The batch process was executed at 4 L working volume in a Sartorius B5vessel. Cells were inoculated at 3.10⁵ cells.mL⁻¹ in VPRO medium (SAFC)supplemented with 6 mM L-glutamin and subsequently cultured for 17 days.

Fed-Batch Process B

The fed-Batch process was executed at 4 L working volume in a SartoriusB5 vessel. Cells were inoculated at 3.10⁵ cells.mL⁻¹ in VPRO medium(SAFC) supplemented with 6 mM L-Glutamin. During the culture glucose andglutamine were added to keep the concentration above respectively 15 mMand 1 mM. Amino Acids and peptides were added from day 5 to replenishthe consumed amino acids.

Process of the Invention C3

The process of the invention was performed in a 2 L Applikon vessel. A100 kDa Molecular Weight Cut-Off (MWCO) hollow fiber membrane (GE)operated in ATF flow mode with an ATF-2 system (Refine Technology) wasused to retain the cells and the IgG product. The culture was startedwith 3×10e5 cells/mL in VPRO medium (SAFC) supplemented with 6 mML-Glutamin. VPRO culture medium (SAFC) supplemented 6 mM L-Glutamin isperfused through the suspension cell culture using an SPR between 0.05and 0.2 nL.cell⁻¹.day⁻¹. Cell culture is removed at 10% of the workingvolume per day above 10.10⁶ cells.mL⁻¹ and at 30% of the working volumeper day when the viable cell density exceeds 30.10⁶ cells.mL⁻¹ andonwards.

Result

As can be seen from FIG. 5 with the process of the invention higherviable cell densities are reached fast, Furthermore, FIG. 5 also showsthat the viability of the cells can be maintained longer with theprocess of the invention as process C3 was maintained in operation overa period of nearly 40 days, because no clogging of the retention deviceoccurred even with high cell densities.

FIG. 6 shows that product concentrations for the process of the presentinvention are much higher than the product concentration in the batchprocess. The product flow containing the product was harvested fromprocess C3 at approximately 200% to 250% times the final concentrationin the batch process A.

FIG. 7 shows that most product is formed by the process of the presentinvention and that the process of the invention can be maintained longerthan the batch process A or the fed-batch B. At day 17, the cumulativeyield of process C3 is 8.1 times the cumulative yield of the batchprocess (A3) and 2.1 times the cumulative yield of the fed-batch process(B). Also, at day 17, the batch process ended. At day 21, the cumulativeyield of process C3 is 3.0 times the cumulative yield of the fed-batchprocess B. At day 21, the fed-batch process ended. After 39 days, theoverall cumulative yield of process n3 is 25 times the cumulative yieldof the batch process A and 6 times the yield of the fed-batch process B.

It can be concluded from this experiment that the overall yield of adesired biological material in the process according to the presentinvention can be further improved by applying a bleed of the cellculture when the cell density exceeds a certain high level.

Example 4 Culturing of and Production with CHO Cells

In this example the process according to the present invention has beenperformed with an IgG producing CHO cell line and includes a temperaturedrop to decrease cell growth.

FIG. 8 shows the cell number Y (10⁶.ml⁻¹) plotted versus the processtime X (days) for C4 (process of the invention).

FIG. 9 shows the IgG concentration in the reactor Z (% as compared tothe maximum IgG concentration reached versus the process time X (days)for process C4 (one embodiment of process of the invention)

The fermentation was performed using a Sartorius Biostat B controller tocontrol temperature at 36.5° C., pH between 7.1 and 6.9 and DO at 40%air saturation and at 100 rpm. The temperature was dropped to 32° C. onday 5.

Process of the Invention C4

The process of the invention was performed in a 2 L Applikon vessel.Cell and product retention device is a 50 kD Molecular Weight Cut-Off(MWCO) hollow fiber membrane (General Electric) operated in ATF flowmode with an ATF-2 system (Refine Technology). The culture was startedwith 5.10⁶ cells.mL⁻¹ in MTCM-49 culture medium (Hyclone). The mediumwas perfused through the suspension cell culture using an SPR between0.1 and 0.4 nL.cell⁻¹.day⁻¹. The CO₂ pressure was controlled below 15%.

Result

The data show that the process of the invention also works when using aprotein producing CHO cell line. The achieved cell density and theproduct concentrations are increased compared to batch culture. The dataalso show that in the process according to the present invention cellgrowth can be arrested (e.,g. by a temperature drop), whereas theproduct accumulation in the culture system continues.

Example 5 Process of the Invention Performed with a Myeloma Cell Line

The process according to the present invention can also be applied tomyeloma cell lines. To this end the fermentation is performed using aSartorius Biostat B controller to control temperature at 36.5° C., pHbetween 7.2 and 6.8 and DO at 40% air saturation and at 100 rpm. Thecell culturing starts with inoculating the myeloma cells at 3×10e5cells/ml in SFM4Mab culture medium (Hyclone) in a 5 L Sartorius vessel.The cell and product retention device is a 30 kD Molecular WeightCut-Off (MWCO) hollow fiber membrane (General Electric) operated in ATFflow mode with an ATF-4 system (Refine Technology). SFM4Mab culturemedium (Hyclone) is perfused through the suspension cell culture usingan SPR between 0.1 and 0.4 nL.cell-1.day-1. The CO2 pressure iscontrolled below 15%.

Example 6 Process of the Invention Performed with an MDCK Cell Line

The process according to the present invention can also be applied totransformed MOCK cell lines in suspension. To this end the fermentationis performed using a Sartorius Biostat B controller to controltemperature at 36.5° C., pH between 7.2 and 6.8 and DO at 40% airsaturation and at 100 rpm. The cell culturing starts with inoculatingthe transformed MDCK cells at 3×10e5 cells/ml in VP-SEM culture medium(Invitrogen) in a 5 L Sartorius vessel. The cell and product retentiondevice is a 30 kD Molecular Weight Cut-Off (MWCO) hollow fiber membrane(General Electric) operated in ATF flow mode with an ATF-4 system(Refine Technology). VP-SFM culture medium (Invitrogen) is perfusedthrough the suspension cell culture using an SPR between 0.1 and 0.4nL.cell-1.day-1. The CO2 pressure is controlled below 15%.

The invention claimed is:
 1. Process for culturing of cells in a reactorin suspension in a cell culture medium, comprising: circulating a cellculture through a separation system comprising a filter, wherein thecell culture comprises eukaryotic cells, a biological substance producedby the cells, and a cell culture medium, wherein at least one cellculture medium component is fed to the cell culture; wherein the cellculture is circulated through the filter in an alternating tangentialflow; and wherein the separation system separates the biologicalsubstance from substances having a lower molecular weight than thebiological substance; retaining or feeding back the biological substanceinto the reactor, wherein the cells are cultured to have a viable celldensity of at least 10×10⁶ cells/mL; and harvesting the biologicalsubstance from the cells and/or from the cell culture; wherein thebiological substance is present in the cell culture at a concentrationof at least 5 g/L prior to the harvesting.
 2. Process according to claim1, wherein the cell culture is continuously circulated through theseparation system, and wherein part of the substances of lower molecularweight is continuously removed from the cell culture.
 3. Processaccording to claim 1, wherein the cells are mammalian cells.
 4. Processaccording to claim 1, wherein the biological substance is a protein. 5.Process according to claim 1, wherein the separation system is a hollowfiber filter.
 6. Process according to claim 1 wherein cell culture isremoved at least once from the reactor and cell culture medium is addedto the reactor to compensate for the cell culture removal.
 7. Processaccording to claim 1, wherein cell culture conditions are chosen suchthat cell growth rate and/or specific productivity of the cells is notlimited.
 8. Process according to claim 7, wherein the cell cultureconditions are chosen such that the concentration of at least one of thecomponents of the cell culture medium remains constant.
 9. Processaccording to claim 1, wherein at least the depleted nutrients aresupplemented by feeding these nutrients to the reactor.
 10. The processof claim 1, wherein the biological substance is present in the cellculture at a concentration of at least 10 g/L prior to the harvesting.11. The process of claim 10, wherein the cells are cultured to have aviable cell density of at least 50×10⁶ cells/mL.
 12. The process ofclaim 4, wherein the harvesting comprises harvesting the protein fromthe flow which contents are kept in or fed back into the reactor, fromcell culture that is removed from the reactor, or both.
 13. The processof claim 12, wherein the harvesting comprises subjecting the flow whichcontents are kept in or fed back into the reactor, the cell culture thatis removed from the reactor, or both to affinity chromatography and/orion exchange chromatography.
 14. The process of claim 12, wherein theprotein is present in the cell culture at a concentration of at least 10g/L prior to the harvesting.